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MONITORING ESSENTIAL OIL QUALITY OF ETLINGERA SP1 BY
GC AND GC-MS
SIVANESAN SHANMUGAM
A thesis submitted in fulfillment of the requirements for the award of thedegree
of Bachelor of Chemical Engineering
Faculty of Chemical & Natural Resources Engineering
Universiti Malaysia Pahang
MAY 2008
ii
I declare that this thesis entitled “Monitoring Essential Oil Quality of Etlingera sp1
BY GC and GC-MS” is the result of my own research except as cited in the
references. The thesis has not been accepted for any degree and is not concurrently
submitted in candidature of any other degree.
Signature : …………………….........
Name of Candidate : …………………............
Date : ………………................
iii
DEDICATION
Specially dedicated to my late grandmother, family and friends for their love and
care…
iv
ACKNOWLEDEGMENT
Completion and submission of a thesis of this kind involves lots of hard work
and sacrifices. This thesis is a result of almost a year of study whereby I have been
accompanied and fostered by many. It is a pleasant moment that I have now the
opportunity to express my gratitude to all of them.
Firstly I thank God for His blessing that I finally able to complete my thesis.
Secondly, I would like to thank my supervisor, Prof. Dr. Mashitah Mohd. Yusoff
Almost a year I have known Dr. Mashitah as a principle-centered person. Her
enthusiasm and integral view on chemistry and research has made a deep impression
on me. I would like to thank her as she always kept an eye on the progress of my
research in addition to her busy schedule. She has been a very handful person for me
to complete my thesis successfully.
At the same time, I would like to thank Cik Nurulhusna Hamid of Institute of
Medical Research who guided me patiently throughout data collection and analysis.
In particular, I also like to extend my thanking to all my colleagues and others
that have assisted me in various occasions. Unfortunately, I am afraid that I cannot
list all of them in this limited space. Foremost I am thanking for having a supportive
family and friends that boosted me when I was down in the mid of the study. Thank
you for being there.
v
ABSTRACT
Etlingera sp.1 of the family Zingiberaceae is being studied for changes in its
essential oil quality during storage. Zingiberaceae comprises about 1200 species of
which 1000 is distributed throughout Tropical Asia. Essential oil exists in all species
in the genus Etlingera. The highest content is usually in the rhizomes of this plant.
Essential oil extracted from the rhizomes is primarily used in fragrance making and
have high commercial value due to its antibacterial and therapeutic properties.
Suitable extraction method needs to be chosen as essential oils are composed of heat-
sensitive chemical constituents. In this study, the Clevenger-type hydrodistillation
method was employed due to ease of use and milder extracting condition. Essential
oil composition was analyzed using GC and GC-MS. Generally, gas
chromatographic techniques are used to separate mixtures of chemical constituents
into individual components Compounds present were identified using GC-MS while
GC was used for weekly analysis. n-Hexane was chosen as solvent as previous
studies using n-hexane reportedly produced optimum results. Comparative analysis
conducted over a three weeks period indicated that the quality of essential oil
exposed to light and temperature underwent minor changes.
vi
ABSTRAK
Etlingera sp.1 dari famili Zingiberaceae telah dikaji perubahan ke atas kualiti
minyak patinya semasa penyimpanan. Zingiberaceae terdiri daripada 1200 spesis
dengan seribu darinya tertabur di keseluruhan Asia Tropika. Minyak pati wujud di
semua spesis dalam genus Etlingera. Kandungan tertinggi minyak pati ialah di dalam
rizom tumbuhan. Minyak pati yang diekstrak daripada rizom digunakan di dalam
persediaan fragran dan mempunyai nilai kormesial tinggi sebagai antibakteria dan
terapeutik. Kaedah pengekstrakan yang sesuai adalah perlu disebabkan komposisi
minyak pati adalah sensitif haba. Di dalam kajian ini, metod penyulingan hidro jenis
Clevenger digunakan kerana mudah dan kondisi pengekstrakannya yang sederhana.
Komposisi minyak pati dianalisa menggunakan GC dan GC-MS. Amnya, teknik
kromatografi gas diguna untuk memisahkan campuran kimia kepada kompaun
individu. GC-MS diguna untuk mengenalpasti kompaun yang hadir dalam sampel
manakala GC diguna untuk analisis mingguan. n-Heksana dipilih sebagai pelarut
memandangkan kajian-kajian sebelumnya melaporkan hasil yang optimum dengan
penggunaannya. Perbandingan analisis yang dibuat selama tiga minggu mendapati
kualiti minyak pati telah mengalami perubahan kecil-kecilan.
vii
TABLE OF CONTENTS
CHAPTER TITLE PAGE
TITLE PAGE i
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF APPENDICES xiii
LIST OF ABBREVIATIONS xiv
1 INTRODUCTION
1.1 Background 1
1.2 Problem statement 1
1.3 Objective 2
1.4 Research scope 2
viii
2 LITERATURE REVIEW
2.1 The Genus Etlingera - An overview 3
2.1.1 Ecology 4
2.1.2 Medicine 5
2.1.3 Selected plants of the genus Etlingera 5
2.1.4 Essential oil 8
2.1.5 Gas Chromatography 10
2.1.5.1 Recorders 13
2.1.5.2 Retention Time 13
2.1.5.3 Factors which affect GC 13
2.1.5.4 Mass spectroscopy 14
2.1.6 Applications of GC-MS 15
2.1.7 Distillation methods 16
2.1.7.1 Steam distillation 16
2.1.7.2 Hydrodistillation 17
2.1.7.3 Water & steam distillation 18
2.1.7.4 Solvent Extraction 19
2.1.7.5 Carbon Dioxide Extraction 19
2.1.7.6 Cold Pressing 20
2.1.7.7 Florasols/ Phytols 20
3 METERIAL AND METHODS
3.1 Introduction 21
3.2 Sample Preparation 22
3.3 Isolation of the Essential Oil 23
3.4 Sample Analysis 23
3.4.1 GC-MS Analysis (operating conditions) 23
3.4.2 GC Analysis (operating conditions) 24
ix
4 RESULTS AND DISCUSSION
4.1 Results 25
4.1.1 Yield of Product 25
4.1.2 Physical characteristics of Product 26
4.1.3 Gas Chromatography 26
4.1.3.1 GC-MS Results 28
4.1.3.2 GC Results 31
4.1.4 Chromatogram Comparison Analysis 34
4.1.4.1 Comparison for Light Test 36
4.1.4.2 Comparison for Temp Test 37
4.1.5 Compounds Identification 38
4.1.6 Decanoic Acid 38
4.1.7 Dodecanoic Acid 39
4.1.8 n-Hexadecanoic Acid 40
5 CONCLUSION & RECOMMENDATION
5.1 Conclusion 41
5.2 Recommendations 41
REFERENCES 42
APPENDICES 45
x
LIST OF TABLES
TABLE NO TITLE PAGE
2.1 Boiling points of some common essential oilcomponents, °C
18
4.1 Yield of Essential Oil 25
4.2 Product physical characteristics 26
4.3 Fresh Essential Oil peak area 35
4.4 Light test peak area 35
4.5 Temperature test peak area 36
4.6 Summary of compound properties 40
xi
LIST OF FIGURES
FIGURE NO TITLE PAGE
2.1 Taxonomy of Ginger (Farlex, 2004) 3
2.2 Etlingera Elatior “Yamamoto”(http://www.alohatropicals.com )
5
2.3 Etlingera Elatior “Thai Queen”(http://www.alohatropicals.com )
6
2.4 Etlingera hemisphaerica “Helani Tulip”(http://www.alohatropicals.com)
6
2.5 Etlingera Elatior “PinkTorch Ginger”( http://www.alohatropicals.com )
6
2.6 Etlingera Elatior “Red Torch Ginger”(http://www.alohatropicals.com )
6
2.7 Etlingera megalocheilos, Keningau, Sabah (Axel, 2007) 7
2.8 Etlingera brevilabrum, Maliau Basin, Sabah (Axel,2007)
7
2.9 Etlingera australasica, Daintree, North Queensland(Axel, 2007)
7
2.10 Etlingera coccinea, Bario, Sarawak (Axel, 2007) 7
2.11 Etlingera velutina, Sabah (Axel, 2007) 8
2.12 Etlingera venusta “Malay rose” (Axel, 2007) 8
2.13 Essential Oil extraction chart (www.healing.about.com) 9
2.14 Plant bases of various Essential Oils(www.healing.about.com)
10
2.15 Gas Chromatography(http://www.j-chrom-sci.com, 2007)
11
2.16 Simplified Gas Chromatography Parts(http://www.j-chrom-sci.com, 2007)
11
2.17 Parts in Gas Chromatography 12
2.18 Clevenger Type Hydrodistillation apparatus(http://heartmagic.com.eooptions)
17
3.1 Summary of Methodology 22
4.1 Light test samples 26
4.2 Temperature test samples 27
4.3 Chromatogram and data of 5.973 min 28
xii
4.4 Chromatogram and data of 7.548 min 29
4.5 Chromatogram and data of 10.353 min 30
4.6 GC-FID analysis for fresh Etlingera sp.1 essential oil 31
4.7 GC-FID analysis for test of temperature week 1 31
4.8 GC-FID analysis for test of temperature week 2 32
4.9 GC-FID analysis for test of temperature week 3 32
4.10 GC-FID analysis for test of light week 1 33
4.11 GC-FID analysis for test of light week 2 33
4.12 GC-FID analysis for test of light week 3 34
4.13 Decanoic acid C10H20O2 38
4.14 Dodecanoic acid CH3(CH2)10COOH 39
4.15 n-Hexadecanoic acid C16H32O2 40
xiv
LIST OF ABBREVATIONS AND SYMBOLS
T = Temperature
t = Time
cm = Centimeter
m = Meter
g = Gram
C = Degree Celcius
K = Kelvin
% = Percentage
g/mL = gram per milliliter
mg/L = Milligram per litre
rpm = rotation per minute
xiii
LIST OF APPENDICES
APPENDIX APPENDIX PAGE
A APPENDIX A (GC-MS Datasheet) 45
B APPENDIX B (GC-FID Datasheet) 52
C APPENDIX C (Apparatus) 67
CHAPTER 1
INTRODUCTION
1.1 Background
Zingiberaceae, or the family of ginger in layman term, is a family of flowering
plants consisting of aromatic lasting herbs with rhizomes, comprising 52 genera and more
than 1300 species, distributed throughout tropical Africa, Asia and the Americas
(Wikipedia, 2007).The most popular species from this family is the edible ginger of
commerce known in Malay as “halia” (scientific name: Zingiber officinale).Noticeably,
from its total population, around 1000 species occur in tropical Asia.Many species are
important ornamental plants, spices, or medicinal plants. Attractive genera include the
shell gingers (Alpinia), Siam or summer tulip (Curcuma alismatifolia), Globba, ginger
lily (Hedychium), Kaempferia , torch-ginger Nicolaia, Renealmia, and ginger (Zingiber).
Spices include ginger (Zingiber), galangal or Thai ginger (Alpinia galanga and others),
melegueta pepper (Aframomum melegueta), myoga (Zingiber mioga), turmeric
(Curcuma), cardamom (Amomum, Elettaria).
1.2 Problem Statement
Quality of essential oils degrades easily upon exposure to light, air and changing
climate. The study is to monitor changes in the essential oil of an Etlingera species using
2
GC-FID and GC-MS under different conditions, namely, temperature and light, over
time.
1.3 Objectives
The objective of this study is to perform a comparison study between samples of
essential oil obtained from Etlingera sp.1.The aim of this experiment is to monitor the
changes in essential oil quality Etlingera sp.1 using GC-FID and GC-MS upon certain
length of time to heat and light and to identify the compounds present.
1.4 Research Scope
Variation in Etlingera sp.1 essential oil sample is being studied for its property
changes over 3 weeks period.
CHAPTER 2
LITERATURE REVIEW
2.1 The Genus Etlingera - An overview
Kingdom PlantaeSubkingdom Viridaeplantae
Phylum TracheophytaSubphylum SpermatophytinaIntraphylum Angiosperma
Division MagnoliphytaClass LiliopsidaOrder Zingiberales
Family ZingiberaceceaeGenus Etlingera
Species sp.1Scientific name ?Common name ?
Figure 2.1: Taxonomy of Ginger (Farlex, 2004)
The genus Etlingera is distributed from India to the Pacific Islands with Borneo
and New Guinea seem to be the most popular location of this species. To date, botanists
estimate about 10 species of Etlingera distributed throughout Peninsular Malaysia and
Singapore. The varying shades of pink and red colours of bracts and flowers make
Etlingera species very attractive plants. Plants of Etlingera have various traditional and
commercial uses. While comprehensive morphological examination is being carried out
by the botanist at Universiti Malaya, the specimen that used is this study will be named
Etlingera sp. 1
4
Even though we do not have specific information on this species, we can expect
similarities from previous researches done on the other species which belong to the same
genus, Etlingera. For example, In Sabah, Malaysia, the hearts of young shoots, flower
buds, and fruits of E. elatior, E. rubrolutea, and E. littoralis are consumed by indigenous
communities as condiment, eaten raw or cooked (Noweg, Abdullah, & Nidang, 2003). In
Thailand, fruits and cores of young stems of E. littoralis are edible, and flowers of E.
maingayi are eaten as vegetables (Sirirugsa, 1999). Inflorescences of E. elatior are widely
cultivated throughout the tropics as spices for food flavoring and as ornamentals. They
are commonly used as ingredients of dishes such as laksa asam, nasi kerabu, and nasi
ulam in Peninsular Malaysia (Larsen, Ibrahim, Khaw, & Saw 1999). Farms in Australia
and Costa Rica are cultivating the species and selling its inflorescences as cut flowers
(Larsen et al., 1999). In Malaysia, fruits of E. elatior are used traditionally to treat
earache, while leaves are applied for cleaning wounds (Ibrahim & Setyowati, 1999).
Leaves of E. elatior, mixed with other aromatic herbs in water, are used by post-partum
(depression after pregnancy) women for bathing to remove body odor. Phytochemical
studies on rhizomes of E. elatior led to the isolation of two new and six known
compounds of diarylheptanoids, labdane diterpenoids, and steroids (Habsah et al., 2005).
Ethanolic extracts from the flower shoots of E. elatior have antimicrobial activity and are
cytotoxic to heal cells (Mackeen et al., 1997).Past studies on the antioxidant activity of
ginger species were confined to rhizomes (Jitoe et al., 1992; Habsah et al., 2000;
Zaeoung, Plubrukarn, & Keawpradub, 2005). Their rhizomes have been reported to
contain antioxidants. (Chan et al., 2007)
2.1.1 Ecology
Species of Etlingera can be more than 5 m tall and become dominant in gaps (E.
megalocheilos, E. brevilabrum, E. coccinea, E. fimbriobracteata).The reproductive
5
biology has been investigated by Ms. Louise Pedersen (Univ. Copenhagen) who found
that spider hunters are important pollinators (Axel, 2007).
2.1.2 Medicine
The species sp.1 that is being studied is likely to have medicinal and therapeutic
properties just like other members of the Zingiberaceae family. Many previous studies
have proved this to be true. An organic extract of Etlingera aff. rosea B.L. Burtt & R.M.
Sm. (Zingiberaceae) was found to exhibit significant cytotoxic activity when evaluated
against a panel of human cancer cell lines. Leaves of Etlingera species inhibited Gram-
positive bacteria. With promising antioxidant and antibacterial properties, leaves of
Etlingera species have great potential to be developed into natural preservatives and
herbal products, applicable to the food and pharmaceutical industries. Unlike the
commercial use of rhizomes, the harvesting of leaves does not result in destructive
sampling of plants (Chan et al, 2007).
2.1.3 Selected plants from the genera Etlingera
Figure 2.2: Etlingera Elatior “Yamamoto” (http://www.alohatropicals.com )
6
Figure 2.3: Etlingera Elatior “Thai Queen” (http://www.alohatropicals.com )
Figure 2.4: Etlingera hemisphaerica “Helani Tulip” (http://www.alohatropicals.com)
Figure 2.5: Etlingera Elatior “PinkTorch Ginger”( http://www.alohatropicals.com )
Figure 2.6: Etlingera Elatior “Red Torch Ginger” (http://www.alohatropicals.com )
7
Figure 2.7: Etlingera megalocheilos, Keningau, Sabah (Axel, 2007)
Figure 2.8: Etlingera brevilabrum, Maliau Basin, Sabah (Axel, 2007)
Figure 2.9: Etlingera australasica, Daintree, North Queensland (Axel, 2007)
Figure 2.10: Etlingera coccinea, Bario, Sarawak (Axel, 2007)
8
Figure 2.11: Etlingera velutina, Sabah (Axel, 2007)
Figure 2.12: Etlingera venusta “malay rose” (Axel, 2007)
2.1.4 Essential oil
The essential oil of a material is the name given to the mixtures of substances
extracted from a biological system and contains the essential components that provide the
characteristic smell or flavor of that material. They are also known as volatile or refined
oils or simply as the oil of the plant material from which they were extracted. The term
essential indicates that the oil carries distinctive fragrance (essence) of the plant,
importantly or core substance.For example, peppermint oil, patchouli oil, jasmine oil and
etc. Essential oils are usually a highly complex mixture of a wide variety of substances.
9
The oil may often be simulated by a fairly simple mixture of artificial compounds
blended in the same percentage as the original oil but the aroma or taste often lacks the
validity of the original essential oil (Yoshiro, 1976). Essential oils are extracted by
different methods. The material is sometimes leached with water and the oil steam
distilled from the aqueous mixture. The natural material may also be solvent extracted
and the oil recovered by distillation. Extraction must be done with care as many of the
components of essential oils are temperature sensitive. Essential oils analysis without the
use of gas chromatography would be extremely difficult. Prior to the technique being
developed, only the major components of the oils could be separated, achieved by
distillation with high efficiency columns (Holttum, 1965).
Figure 2.13: Essential Oil extraction chart (www.healing.about.com)
The extraction method depends on the plant material as well as the type of end
product that is desired. Essential oils been used medicinally at different periods in the
past. Medical applications proposed by those who sell medicinal oils vary from skin
treatments to remedies for cancer, and are often based on historical use of these oils for
these purposes.
10
Interest in essential oils has revived in recent times, with the popularity of
aromatherapy, a branch of alternative medicine which claims that the specific aromas
carried by essential oils have curative effects.
Berries
Allspice Juniper
Seeds
Almond Anise Nutmeg oil
Wood
Camphor Cedar Rosewood Sandalwood
Rhizome
Ginger
Leaves
Basil Bay leaf Cinnamon Common sage Eucalyptus Lemon grass Oregano Patchouli Peppermint Pine Rosemary Spearmint
Resin
Frankincense Myrrh
Flowers
Chamomile Clary sage Clove Geranium Hyssop Jasmine Lavender Marjoram Orange Rose Ylang-ylang
Peel
Bergamot Lime Orange Tangerine
Root
Valerian
Figure 2.14: Plant bases of various Essential Oils (www.healing.about.com)
